A novel family of DNA mismatch-specific endonucleases from plants was discovered recently (Oleykowski, et al. (1998) Nucl. Acid Res. 26:4597-4602; Yang, et al. (2000) Biochem. 39:3533-3541). The plant source with the highest apparent concentration of this class of endonucleases is celery (Oleykowski, et al. (1998) supra), and thus the enzyme was purified from celery and named CEL I (Oleykowski, et al. (1998) supra; Yang, et al. (2000) supra). CEL I cleaves DNA at the 3′-side of sites of base-substitution mismatch and DNA distortion (Oleykowski, et al. (1998) supra; Yang, et al. (2000) supra).
CEL I has been shown to be useful in mismatch detection assays that rely on nicking or cleaving duplex DNA at insertion/deletion and base-substitution mismatches (Oleykowski, et al. (1998) supra; Yang, et al. (2000) supra; Kulinski, et al. (2000) BioTechniques 29:44-48; Colbert, et al. (20001) Plant Physiol. 126:480-484; Sokurenko, et al. (2001) Nucl. Acids Res. 29:e111; U.S. Pat. No. 5,869,245).
Purified preparations of CEL nuclease identified as CEL I contain two different protein species, CEL I and CEL II (Yang, et al. (2000) supra; U.S. Pat. No. 5,869,245). One species, called CEL I, has an apparent molecular weight of 43 kDa as determined by SDS-PAGE. Removal of N-linked oligosaccharides with Endo Hf reduces the molecular weight to 29 kDa. CEL I was partially sequenced and the gene encoding CEL I was isolated from a celery cDNA library, sequenced, and cloned into E. coli (Yang, et al. (2000) supra; U.S. Pat. No. 5,869,245). CEL II has an apparent molecular weight of 39 kDa as determined by SDS-PAGE and removal of N-linked oligosaccharides reduces the molecular weight to 37 kDa. Chromatographic separation of CEL I and CEL II has been described and while CEL I and CEL II appear to be related, they have different enzymatic activity; CEL II has a higher pH optimum than CEL I and CEL II is more efficient than CEL I in cleaving DNA at mismatches.
Therefore, needed in the art is a readily available source of isolated CEL II, which lacks CEL I contamination, for use in detecting the presence of mismatches in double-stranded DNA. The present invention meets this long-felt need in providing nucleic acid sequences encoding CEL II for recombinant production of the same.